Week 14
Thank you Hailey - Dr. B 121714 Since the last samples did not have any working enzyme, 2 L or 4 500 mL of LB were expressed and spun down. They were resuspended in lysis buffer and stored in the -80 degree Celsius until next semester. Hopefully a working enzyme will be purified for next semester so enzyme assays can continue and inhibition assays can begin
12042014- Great job 1162014- Include more pictures and explanations
11-13 Week
Analysis: We ran all of our samples at least once if not multiple times, the samples from our expressions were inactive. We need to re-express and purify to begin the enzyme assays. We will probably run future enzyme assays at trial 6 concentrations since it was 4x the Km values for each substrate and hopefully we will get functional enzyme as our inhibitors have arrived.
Fig 7) Trial 7 increased working dilutions to 0.5 and decreased total volume to 400 uL so more likely to collide with substrates and enzymes. Used sample 1 (glycerol), initial climb is adding NADPH(30 uL), then second with 200 uL of sodium acetate buffer climbed to 2 absorbance. The second peak is adding Biopterin (30 uL), the next peak in was addition of 50 uL of enzyme, the next peak was an additional 150 uL of enzyme then an additional 40 uL of enzyme. Since absorbance was constant the enzyme was inactive(or we had reached too high absorbance and the photospectometer couldn't detect reliably).
Fig 6) Trial 6 increased working dilutions to 0.5 and decreased total volume to 400 uL so more likely to collide with substrates and enzymes. Used sample 1 (glycerol), initial climb is adding NADPH(20 uL), then second climb to 1.5 absorbance is 10 uL with 200 uL of sodium acetate buffer. The second peak is adding Biopterin (30 uL), the next peak in was addition of 50 uL of enzyme, the next peak was an additional 50 uL of enzyme. Since absorbance kept increasing due to enzyme, something contaminating the enzyme was contributing to absorbance.
Fig 5) Trial 5 with sample 2 (snap frozen), initial climb is adding NADPH(180 uL) with 200 uL of sodium acetate buffer. The second peak is adding Biopterin (180 uL), the next fall around 8 minutes in was addition of 50 uL of enzyme the next peak was an additional 50 uL of enzyme, the next peak was 40 uL of enzyme. No drops thus concluded enzyme inactive, added 20% glycerol and stored in -20 C.
Fig 4 Trial 4 with sample 1 (glycerol), first peak is adding NADPH(180 uL) with 200 uL of sodium acetate buffer. The downward peak is adding Biopterin (180 uL), the next upward peak and subsequent drop was 50 uL of enzyme added. The next drop was 50 uL of buffer, since absorbance went down then supported that drops in absorbance were caused by decreasing concentration of NADPH by adding volume rather than enzyme activity.Enzyme concluded to be inactive.
Fig 3) Trial 3 with sample 2 (glycerol), first peak is adding NADPH(150 uL) with 200 uL of sodium acetate buffer, then second peak and climb to around 7 minutes is adding more NADPH until the total NADPH was 200 uL of NADPH. The peak around 8 min. is adding Biopterin (200 uL), the rest of the peaks both up and down were adding enzyme until the final added was 100 uL. Abnormal since absorbance should be going up or down.Concluded enzyme sample was inactive.
Fig 2) Trial 2 with sample 2 (glycerol), initial climb is adding NADPH(180 uL) with 200 uL of sodium acetate buffer. The second peak is adding Biopterin (180 uL), the next fall around 8 minutes in was addition of 50 uL of enzyme the next fall was an additional 50 uL of enzyme. Initially thought drops were due to enzyme activity being very fast so that absorbance dropped drastically. Later proven otherwise in trial 4.
Fig 1) Trial 1 Assay 1 with sample 1 (glycerol), first peak is adding NADPH(30 uL) with 200 uL of sodium acetate buffer. The second peak is adding Biopterin (30 uL), the rest of the peaks are adding enzyme until the final added was 100 uL. Abnormal since absorbance should be going up or down. Originally thought it was machine error until trial 6. Later in trial 6 it seems like there was contamination in the sample that added absorbance.
Worked on Enzyme assays stored 6- Biopterin in 0.5 MNaOH in 50 uL aliquots and NADPH in 10mM NaOH both stored in -80 C. Began Enzyme Assays on the 2 expression runs stored in 20% glycerol and snap frozen. Sample 1(method of storage) will refer to the first expression 1 in a specific storage in the discussion below. The assay used was a photometric assay to measure the disappearance of NADPH at 340 nM. Working solutions were a concentration of 0.2 M for both NADPH and Biopterin.
Weeks nine and ten
Ran DVP and ordered 2 additional compounds. Began another round of expression for round 3.
ACL2449_Elut1_102914.jpg
Fig 1) Elution 1 concentration of 4.20 mg/mL need to characterize and FPLC.
ACL2449_Elut2_1029.jpg
Fig 2) Elution 2 concentration 0.45 mg/mL need to characterize and FPLC.
Will probably run both elutions in FPLC since that gives a higher final concentration. Seventh and Eight Weeks Fig 1) Results of FPLC from fifth and sixth week. We collected tubes 41-54. The size is around 60 kDa thus the protein most likely dimerized. We stored 1 mL of 0.7 mg/mL in 20% glycerol and another 1 mL of 0.78 mg/mL sample in liquid nitrogen(will retrieve the nanodrop graphs when I return to lab)
We began another round of expression with a consistent salt gradient of around 900 mM for the entire process. We need to run an SDS page gel for our sample and do FPLC. Our goal is to get at least one sample of enzyme that is not a dimer to see if dimerizing affects the activity and or inhibition of our enzyme.
We ran our first library of ~30,000 compounds using the NAPD site as our ligand. After our second run, we will have another run with our other substrate DVP(using a dummy ligand) to run another virtual screening. We will most likely select some ligands from both runs and see which site has the most inhibition. We are also planning our enzyme assay, we will measure the absorbance of NADP at around 520 nM. If the enzyme is functioning the absorbance will decrease, if inhibited the absorbance will not change. The Km value for our enzyme for NADP is 0.012 mM. Here is general information about our enzyme:
Fifth and Sixth WeekAfter transforming we did expression in 2 L or 4 times the amount we did last round of expression so we would have extra protein or at least a higher concentration. We then harvested the cells through sonication and spindown. Last expression round we were concerned that PTR1 dimerized since the FPLC machine mainly had a 60kDa protein while our protein is 30 kDa. To try and avoid dimerization this round, we had a salt gradient of 4 samples. Tube A had a salt concentration of 500 mM, tube B a salt concentration of 750 mM, tube C a salt concentration of 1000 mM and tube D a salt concentration of 400 mM. The salt should ideally prevent interactions between two protein molecules and prevent dimerization. We then ran and two SDS characterization gels of the highest salt concentration and the lowest salt concentration samples C(900 mM) and D(400mM). Assuming everything is the same, if samples C and D had not dimerized then neither would the other samples with salt concentrations between C and D. After SDS we plan on doing FPLC to further purify our protein and ensure it has not dimerized. We have already started on virtual screening.
Fig 5) SDS Page Gel protein characterization for sample C or PTR1 sample with 900 mM NaCl. The banding in elutions 1 and 2 appears to be around 35 kDa which is the right size for PTR1. It does not appear to have dimerized and there appears to be little contaimination. We will run FPLC to further purify.
As labeled above: 1 kDa is a 1kDa protein ladder.0 Sample 0(cell lysate before induction)1 Sample 1(cell lysate after induction) 2C Sample 2(Soluble fraction)for sample C3D Sample 3(Flow through) for sample D4C Sample 4(Wash) for sample C5C Sample 5(elution 1) for sample C 6C Sample 6(elution2) for sample C3C Sample 3(flow through) for sample C Fig 4) SDS Page Gel protein characterization for sample C or PTR1 sample with 400 mM NaCl. The banding in elutions 1 and 2 appears to be around 35 kDa which is the right size for PTR1. It does not appear to have dimerized and there appears to be little contaimination. We will run FPLC to further purify. Since neither this sample nor the one in Fig 5 appear to have dimerized it seems likely that sampels A and B will also not have dimerized. As labeled above: 1 kDa is a 1kDa protein ladder.0 Sample 0(cell lysate before induction)1 Sample 1(cell lysate after induction) 2D Sample 2(Soluble fraction)for sample D3D Sample 3(Flow through) for sample D4D Sample 4(Wash) for sample D5D Sample 5(elution 1) for sample D 6D Sample 6(elution2) for sample D
Fig 3) Kan+ Suc agar plates with BL21 cells transformed with 50 ng of PNIC+PTR1 plasmid.
Fig 2) Kan+ Suc agar plates with BL21 cells transformed with 25 ng of PNIC+PTR1 plasmid.
Fig 1) Kan+ Suc agar plates with BL21 cells transformed with 5 ng of PNIC+PTR1 plasmid.
Due to contaimination in the 4 degree fridge we retransformed BL21 cells with PNIC+PTR1. Third and Fourth Week of Fall We grew up the transformed BL21 cells from the second week and performed protein expression. We then used chromatography/binding assay with nickel to further purify the protein and took samples for an SDS page gel. Fig 1) Nanodrop for elution 1(protein) during purification concentration of 1.4 mg/mL measured at wavelength 280 nM. Fig 2) SDS-page gel ran for our protein Pteridine reductase from L. Major. We failed to get a sample 0, but the samples are as follow: Well 3-Cell lysate after induction(sample 1)Well 4-Soluble fraction(sample 2)Well 5-Flow through(sample 3)Well 6-Wash(sample 4)Well 7 -Elution 1(sample 5)Well 8-Elution 2(sample 6) Well 2:ladderWell 3:Sample 1(cell lysate after induction)Well 4: Sample 2(Soluble fraction)Well 5: Sample 3(Flow through)Well 6: Sample 4(Wash)Well 7:Sample 5(elution 1)Well 8: Sample 6(elution2)The bands were around 30 kDa weight which matched our protein, there didn't appear to be a lot of contaimination but we still ran FPLC just to ensure purity. Fig 3) FPLC graph results. We took tubes 28-33 and combined them for our sample. It was odd that the size of our sample(30 kDa) didn't match up with the FPLC machine which was around 50~60 kDa. Fig 4) First attempt at concentrating after FPLC. Nanodroped, total volume of sample was 4mL but too low concentration Fig 5) Second attempt at concentrating further after Fig 4. Nanodrop concentration, total volume of sample was 1 mL, concentration was too low but could not reasonably concentrate it to a lower volume. After concentrating the sample further, we stored .5 mL in 20% glycerol and another .5 mL by snap freezing in liquid nitrogen. We will perform an enzyme assay to see which storage works better, hopefully even with low volume and concentration we will be able to get some indication of which works best. We need to begin virtual screening and restart protein expression. The protein has 3 possible intermembrane regions which would explain the low yields we've been getting after FPLC. Next protein expression we are going to use 2 L of LB to grow up the bacteria which will hopefully have a high enough concentration to move forward. Week 10+11/first and second week of fall Fig 1) DNA sequence forward for sample B1 using T7 primers Fig 2) DNA sequence reverse for sample B1 using T7 primers Fig3) Pairwise alignment of sample B1 and desired DNA sequence(from codon optimized sequence). The N's were the 2% that didn't match, and there were no insertions or deletions. The N's were then confirmed using the chromatograph to be the correct bases. Query= B1 sent in sequence Sbjct=is desired sequence
Fig 4) DNA sequence similarity between B1 and desired DNA sequence. Fig 5) Nanodrop of one clone sample DNA D1 that had a concentration 56.5 ng/uL(sample was sent to DNA sequencing) Fig 6) Nanodrop of one clone sample DNA D2 with a concentration of 32.3 ng/uL it was sent of to DNA sequencing Fig 7) Nanodrop of one clone sample of DNA D3 with concentration of 53.0 ng/uL sent off to DNA sequencing. Fig 8) Nanodrop of Midipreped B1(pNIC + DNA insert) plasmid and got a concentration of around 58.6 ng/uL.
Fig 9) Transformed BL21 cells with clone DNA sample B1 with 1 ng of, 1 colony grown
Fig 10) Transformed BL21 cells with 5 ng clone DNA sample B1 ~1 colony growth Fig 11) Transformed BL21 cells with 25 ng of clone DNA sample B1 a lot of colony growth.*We nanodroped 7 samples about, 3 samples were high enough in concentration to send to DNA sequencing, but only B1 came back as a positive clone. Need to return to lab to get nanodrop pictures. We midipreped an overnight B1 culture, and used the DNA to transform BL21 cells which we will check on Saturday.Week 9Fig 1) Plate with two smears, bottom is A and top is B. A had a ratio of 1 accepting vector to 7 PCR product. B had a ratio of 2 accepting vector to 5 PCR product. Fig 2) Plate with two smears, bottom is C and top is D. C had a ratio of 1 accepting vector to 6 PCR product. D had a ratio of 3 accepting vector to 5 PCR product.
Fig 3) Plate with two smears, bottom is E and top is F. E had a ratio of 4 accepting vector to 7 PCR product. F had a ratio of 1 accepting vector to 9 PCR product. Fig 4) Plate with two smears, bottom is G and top is H. Top had a ratio of 2 accepting vector to 7 PCR product. Bottom had a ratio of 1 accepting vector to 10 PCR product. Fig 5) Plate with two smears, bottom is I and top is J. A had a ratio of 1 accepting vector to 7 PCR product. B had a ratio of 2 accepting vector to 5 PCR product. Fig 6) Plate with two smears, bottom is A and top is B. A had a ratio of 1 accepting vector to 7 PCR product. B had a ratio of 2 accepting vector to 5 PCR product.
Fig 7) Master plate C, the numbers and colonies are as follow:
1:A
2:B
3:C
4:D
5:E
6:F
7:G
8:H
9:I
Fig 8) Masterplate D, the numbers and colonies are as follow
1J
2K
3L
n/a
n/a
n/a
n/a
n/a
n/a
*The nanodrop concentrations for the past master plates have been remarkably low as in less than 20 usually around 10. I had about two colonies from master plate B that had concentrations of 23.4 and 24.9 ng/uL which was enough to send to sequencing. Also I realized that I've been eluting with too much Tris buffer. I've been following the sigma aldrich instructions and using 100 uL instead of 50 uL from the modified cloning protocol. That would decrease the concentration of the miniprep. Therefore I want to rerun all of the masterplates I've made thus far. So in the fall I should redo about 30(9[masterplate A not shown]+9[Masterplate B not shown]+9[Master plate C shown above]+3[Masterplate D shown above]). Hopefully one will yield a positive clone.
Week 8
Fig 6) Gel electrophoresis of PCR squared. 1st well is a 1 kb ladder, the rest of the wells are tubes of PCR squared. Wells 2-5 were concentrated to 208.6 ng/uL(shown in fig. 4) and wells 5-7 were concentrated into 103.1ng/uL(shown in fig 5). Worried about contamination getting for using these samples in ligation independent cloning. Since vectors are more likely to accept smaller DNA bands. Using these samples for initial cloning however, and going to make another PCR squared and more plates. F F
Fig 5) nanodrop concentration for PCR cleanup for the target(a different sample than fig 4 as a backup).
Fig 4) Nanodrop concentration of PCR cleanup for the target. Fig 3) 7.21.14 Gel electrophoresis of PNIC digested by BSAI. Two bands show that plasmid was digested properly and can be used as an accepting vector. Fig 2) 7.18.14 Nanodrop of PCR clean up of target gene. 77.9 ng/uL is a good concentration to continue with cloning Fig 1) 7.20.14 Digested accepting vector for PNIC nanodrop concentration. It is a bit low, but can continue.
Hailey - ok good work. put some dates on these gels (above or below them) Also, don't start the caption with 'this is' - just put 'Agaros Gel of PCR......"Have you started the cloning steps. If not be sure you have enought pNIC-Bsa4. - Dr. B 072114 Fig 1) This is a gel of PCR squared first and last well has the 1 kb ladder. The second well is empty, the rest of the wells have the PCR squared reaction. Wells three and four are not as bright so I did not use them in PCR cleanup. I used wells 5 through 8 in PCR clean-up. The Size is around 900 kb which is the right size for the gene. I need to nanodrop this again next week since I forgot to email myself the graphs but the concentration was around 71 ng/uL for the PCR cleanup.
Fig 2) This a gel of secondary PCR with a temperature gradient. The 1st well has the 1 kb ladder. All of the samples had the same amount of Primary PCR(2 uL), 1 uL of forward primer, 1 uL of reverse primer, 10uL of Reaction buffer, 0.5 uL of Q5 Polymerase(diluted 1:2 from stock) and 5 uL of dNTPs. The protocol differed from the standard Q5 protocol in that the elongation time was extended to 40 seconds. The second well had an anealing temperature of 72 C, the third well 71.6 C, the forth 70.7 C, the fith 67.2C, the sixth 65.6 C, 64.6 C and the seventh 64 C. The brightest band was the fifth well or the well with the annealing temperature of 64.6 C which is the annealing temperature I'll use for PCR squared.
Fig 3) Redid primary PCR for target. The 1st well is the 1 kb ladder, well 2 and 3 are primary PCRs. Since they've formed streaks I used them in Fig 2 for secondary PCR
Also in wet lab we digested PNIC and started growing PNIC bacteria to make an accepting vector. I need to nanodrop it next week in addition to the DNA. Congrats on your secondary working. Include a virtual gel for your RE Digest so we can compare what it's suppose to be and how it came out. -Grace (7/15/14)= ==
Fig 1) Here is a carry over from last week. I ran this gel on friday for PNIC, the ladder is 100 kb. There are 2 bands in the 2nd and 3rd well .306 ng of DNA, the third well had about 3.06 ng of DNA and the fourth well about 30.6 ng of DNA.
Fig 2) Here is a picture of a restriction enzyme digest we did of pgbr22. The first well is a 1 kb ladder, 2nd well is uncut plasmid of pgrb22, 3rd well is pgbr22 cut with ECOR1, 4th well is pgbr22 cut with PvuII, and the 5th well is pgbr22 cut with both ECOR1 and Pvu II. The 3rd well has one band, the 4th well 2 bands and the 5th 2 bands bright bands and possibly 3 additional bands which might be contamination. Other than that it matched the virtual gel. Fig 3) Here is a picture of the successful run of primary PCR. The 1st well is a 1 kb ladder, and wells 4 and 5 are primary PCR attempts. 4 worked, however 5 did not appear to.
Working on secondary PCR hopefully will have full DNA by end of week(goal *cross fingers*)(Ha. Ha. Ha. No.) - Working on secondary PCR have to remake primary PCR. Remaking oligomix, altered annealing temperature for a gradient between 64-72 C for 8 samples and increased elongation time for 40 seconds based on the Q5 website recommendations for complex sequences. Week 5Hailey - good protein gel image. Not sure but I think if you run it longer you will get better separation. I can sort of see 2 bands there that correspond to the 2 bands in FPLC. Be sure to indicate what ladder you use in lane 1. For the PCR gels - crop them so that there is not so much black space. You can crop them int he imaging software before ever saving the image. Also use the 'invert' to change all the dark to light. Do another attempt of the pNIC PCR. you should be able to get bands in each lane. Also indicate which ladder you use (1kb or 100 bp). -- Dr. BThis week:virtual lab: We designed tail primers for our targets and ordered the primers.Wet lab:
ig 1) Ran a protein characterization for Yop H, the gel was weird in that samples ran from one well to another.Well 1-ladder(protein 1kb ladder)Well 2-Cell lystate before induction(sample 0)Well 3-Cell lysate after induction(sample 1)Well 4-Soluble fraction(sample 2)Well 5-Flow through(sample 3)Well 6-Wash(sample 4)Well 7 -Elution 1(sample 5)Well 8-Elution 2(sample 6) Fig 2) The gel electrophoresis of PCRed pGFP from last week. It's hard to see but the fourth well has one band that is about 400-500 bp based on the ladder. There is only one band so there doesn't appear to be any contamination. Fig 3) Gel electrophoresis of PCRed PNIC the first well was the ladder the second well had about .306 ng of DNA, the third well had about 3.06 ng of DNA and the fourth well about 30.6 ng of DNA.The ladder was 100 bp.
Fig4) Results from the FPLC, we appear to have two proteins the one to the left is about 40 kD and the one to the right is about 30 kD. The one to the right or the 30 kD protein is probably YopH since we know YopH's size. We are going to concentrate it and freeze it with nitrogen.
Week 4
Hailey, ok good work. For your Midiprepped pGFP - did you run int on an agarose gel to see if it was contaminated at all? For your PCR the two bands look sharp and of the same height (which is good) - be sure to indicate on the caption that it is a PCR gel ( I wasn't sure if your were just running plasmid or it was PCR). Your YopH concentrations for the protein expression look good. We should have enough for FPLC. Screenshots of the notebook - are kind of ok for this, because we really just need the absorbance value and the shape of the curve, but I would recomend against it for any gels because you don't have good resolution to see them well. I am looking forward to seeing your gel aftter SDS-PAGE. Thanks Dr. B
(running pGFP gel this week after I PCR it, ran out of time last week) Virtual lab:
We analyzed DNA sequences online and designed and plasmid, then we ran virtual digestion on each plasmid.
Wet lab: Updates for DNA sequencing stuff from last week results and next steps: Fig1) Icmb resubmitted the pGFP sequence, it is better than the last submission but the blast below doesn't identify it as pGFP.
Fig 2) Blast for pGFP sequence to compare it, but no recognition as pGFP. Going to run a PCR on pGFP and then run it on a gel to make sure there is no contamination.
Fig 3) The chromatography for the pmcherry sequence from last week. The sequence still needs to be rerun.
New wet lab stuff Fig 4)PCR gel- Pgbr 22 bands. 1st well was 1 kb ladder, 2nd well was tube A( .3ng of DNA, mastermix and primer), 3rd well was tube B(3 ng DNA, primer and mastermix), 4th well was tube C(30 ng DNA, primer and mastermix) and 5th well was control(no DNA, but primers and mastermix). The wells mainly differed in amount of DNA while all the wells had the same amount of mastermix and the same amount of primers(1 ul of forward and 1 ul of reverse primers). Wells 2,3 and 4 should've had one band each. Wells 3 and 4 were successful but Well 2 didn't show any DNA. The control was not supposed to have any bands so it was successful.
This week we purified our YopH protein, here are the results from the nanodrop for elution 1 and elution 2. We made a protein gel and are going to get the results for our protein characterization next week hopefully.
Fig 5) YopH elution 1 nanodrop concentration was 3.7 mg/ml Fig 6) YopH elution 1 trial 2 nanodrop concentration was 3.68 mg/ml (average for elution 1 was 3.69 mg/ml) Fig 7) YopH elution 2 trial 1 nanodrop concentration was 0. 82 mg/ml Fig 8)
YopH elution 2 trial 2 nanodrop concentration was 0.84 mg/ml (average was 0.83mg/ml for elution 2)
Week 3
Hailey - good work. DNA conc should be 106 ng/ul (nanograms per microliter) units.
The pGFP sequence looks pretty bad. We need to look at the electropherogram and determine if we think there is contamination. A Nanodrop read won't give you that information for this type of contaminatinon.
Also - run this sample of pGFP on a gel (about 300 ng of it) to verify if there are contaminating bands. (can run it on another gel that you are already doing if you want)
Include your protein expression (mention it and then enter the cell pellet weight as the 'result' so far) - Dr. B
We made LB with kanomyocin to grow bacteria(which we spent a lot of time on this week) We grew a plasmid this week in lab using DH4alpha bacteria. We purified pGFP and had an averaged nanodrop concentration of 106 ug/uL. Here are the nanodrop results for the DNA.Fig 1) The nanodrop value for the midiprep for pGFP this was averaged with a second trial to yield 106 ul/uL. We also sent the DNA off for verification to ensure we amplified the correct DNA. Fig 2) There are a lot of N's, which means unknown nucleotides. ICMB is also going to rerun this sequence.
Also for week 1 and 2 the DNA sequencing had a lot of N's throughout the sequence so I resent it in for sequencing. The plasmid I had was pmcherry and I used the M14 reverse primer. Andee has the forward primer on her page.
Fig 3) This is the rerun of pm cherry which is worse than the initial sequencing. ICMB is going to rerun the sequence. The next thing we did this week was protein expression. We first grew BL21 bacteria in LB until it reached a specific concentration and then we introduced IPTG so that the bacteria would produce protein. Our protein was YopH. The next day we further purifed the protein and sonicated it. Then we will hopefully do protein expression next week to verify and post the results. Also we did PCR(polymerase chain reaction) on pGBR22 and will probably run it next week on agarose gel.
Week 1 and 2
This week we did some more virtual screening of proteins that we can target, DNA sequencing, made LB agar and broth plates and transformed bacteria.
I sequenced the DNA primer pmcherry and sent it off.Here is the FASTA for nucleotide sequence for pm cherry using a reverse primer. Here are the blast results, pm cherry shows similarity in DNA sequence with at least 19 other sequences.
I tried to use heat shock to transform DH5alpha cells with plasmid PNIC Bsa4 with a resistance to Kan. The plates are below, I got an invisible bacteria lawn(not really). Fig 1) Plate with Dh5alpha cells, plasmid Bsa4 with resistance to Kan(kanamycin) grown on an LB and Kan media. There was no growth and the concentration of plasmid is unknown due to a labeling error. The possible concentrations are 1 ng, 5ng and 25 ng of DNA. Grown for 24 hrs in 37 C incubator. Fig 2) Plate with Dh5alpha cells, plasmid PNIC-Bsa4 with resistance to Kan(kanamycin) grown on an LB and Kan media. There was no growth and the concentration of plasmid is unknown due to a labeling error. The possible concentrations are 1 ng, 5ng and 25 ng of DNA. Grown for 24 hrs in 37 C incubator. Fig 3)Plate with Dh5alpha cells, plasmid Bsa4 with resistance to Kan(kanamycin) grown on an LB and Kan media. There was no growth and the concentration of plasmid is unknown due to a labeling error. The possible concentrations are 1 ng, 5ng and 25 ng of DNA. Grown for 24 hrs in 37 C incubator. Fig 4) Control Plate with Dh5alpha cells grown on an LB and Kan media. There was no growth and the competant cell concetration was less than the other plates. It was used to see if kanamycin was not denatured or expired. Grown for 24 hrs in 37 C incubator.
Thank you Hailey - Dr. B 121714
Since the last samples did not have any working enzyme, 2 L or 4 500 mL of LB were expressed and spun down. They were resuspended in lysis buffer and stored in the -80 degree Celsius until next semester. Hopefully a working enzyme will be purified for next semester so enzyme assays can continue and inhibition assays can begin
12042014- Great job
1162014- Include more pictures and explanations
11-13 Week
Analysis: We ran all of our samples at least once if not multiple times, the samples from our expressions were inactive. We need to re-express and purify to begin the enzyme assays. We will probably run future enzyme assays at trial 6 concentrations since it was 4x the Km values for each substrate and hopefully we will get functional enzyme as our inhibitors have arrived.
Fig 7) Trial 7 increased working dilutions to 0.5 and decreased total volume to 400 uL so more likely to collide with substrates and enzymes. Used sample 1 (glycerol), initial climb is adding NADPH(30 uL), then second with 200 uL of sodium acetate buffer climbed to 2 absorbance. The second peak is adding Biopterin (30 uL), the next peak in was addition of 50 uL of enzyme, the next peak was an additional 150 uL of enzyme then an additional 40 uL of enzyme. Since absorbance was constant the enzyme was inactive(or we had reached too high absorbance and the photospectometer couldn't detect reliably).
Fig 6) Trial 6 increased working dilutions to 0.5 and decreased total volume to 400 uL so more likely to collide with substrates and enzymes. Used sample 1 (glycerol), initial climb is adding NADPH(20 uL), then second climb to 1.5 absorbance is 10 uL with 200 uL of sodium acetate buffer. The second peak is adding Biopterin (30 uL), the next peak in was addition of 50 uL of enzyme, the next peak was an additional 50 uL of enzyme. Since absorbance kept increasing due to enzyme, something contaminating the enzyme was contributing to absorbance.
Fig 5) Trial 5 with sample 2 (snap frozen), initial climb is adding NADPH(180 uL) with 200 uL of sodium acetate buffer. The second peak is adding Biopterin (180 uL), the next fall around 8 minutes in was addition of 50 uL of enzyme the next peak was an additional 50 uL of enzyme, the next peak was 40 uL of enzyme. No drops thus concluded enzyme inactive, added 20% glycerol and stored in -20 C.
Fig 4 Trial 4 with sample 1 (glycerol), first peak is adding NADPH(180 uL) with 200 uL of sodium acetate buffer. The downward peak is adding Biopterin (180 uL), the next upward peak and subsequent drop was 50 uL of enzyme added. The next drop was 50 uL of buffer, since absorbance went down then supported that drops in absorbance were caused by decreasing concentration of NADPH by adding volume rather than enzyme activity.Enzyme concluded to be inactive.
Fig 3) Trial 3 with sample 2 (glycerol), first peak is adding NADPH(150 uL) with 200 uL of sodium acetate buffer, then second peak and climb to around 7 minutes is adding more NADPH until the total NADPH was 200 uL of NADPH. The peak around 8 min. is adding Biopterin (200 uL), the rest of the peaks both up and down were adding enzyme until the final added was 100 uL. Abnormal since absorbance should be going up or down.Concluded enzyme sample was inactive.
Fig 2) Trial 2 with sample 2 (glycerol), initial climb is adding NADPH(180 uL) with 200 uL of sodium acetate buffer. The second peak is adding Biopterin (180 uL), the next fall around 8 minutes in was addition of 50 uL of enzyme the next fall was an additional 50 uL of enzyme. Initially thought drops were due to enzyme activity being very fast so that absorbance dropped drastically. Later proven otherwise in trial 4.
Fig 1) Trial 1 Assay 1 with sample 1 (glycerol), first peak is adding NADPH(30 uL) with 200 uL of sodium acetate buffer. The second peak is adding Biopterin (30 uL), the rest of the peaks are adding enzyme until the final added was 100 uL. Abnormal since absorbance should be going up or down. Originally thought it was machine error until trial 6. Later in trial 6 it seems like there was contamination in the sample that added absorbance.
Worked on Enzyme assays stored 6- Biopterin in 0.5 MNaOH in 50 uL aliquots and NADPH in 10mM NaOH both stored in -80 C. Began Enzyme Assays on the 2 expression runs stored in 20% glycerol and snap frozen. Sample 1(method of storage) will refer to the first expression 1 in a specific storage in the discussion below. The assay used was a photometric assay to measure the disappearance of NADPH at 340 nM. Working solutions were a concentration of 0.2 M for both NADPH and Biopterin.
Weeks nine and ten
Ran DVP and ordered 2 additional compounds. Began another round of expression for round 3.
Fig 1) Elution 1 concentration of 4.20 mg/mL need to characterize and FPLC.
Fig 2) Elution 2 concentration 0.45 mg/mL need to characterize and FPLC.
Will probably run both elutions in FPLC since that gives a higher final concentration.
Seventh and Eight Weeks
Fig 1) Results of FPLC from fifth and sixth week. We collected tubes 41-54. The size is around 60 kDa thus the protein most likely dimerized. We stored 1 mL of 0.7 mg/mL in 20% glycerol and another 1 mL of 0.78 mg/mL sample in liquid nitrogen(will retrieve the nanodrop graphs when I return to lab)
We began another round of expression with a consistent salt gradient of around 900 mM for the entire process. We need to run an SDS page gel for our sample and do FPLC. Our goal is to get at least one sample of enzyme that is not a dimer to see if dimerizing affects the activity and or inhibition of our enzyme.
We ran our first library of ~30,000 compounds using the NAPD site as our ligand. After our second run, we will have another run with our other substrate DVP(using a dummy ligand) to run another virtual screening. We will most likely select some ligands from both runs and see which site has the most inhibition. We are also planning our enzyme assay, we will measure the absorbance of NADP at around 520 nM. If the enzyme is functioning the absorbance will decrease, if inhibited the absorbance will not change. The Km value for our enzyme for NADP is 0.012 mM. Here is general information about our enzyme:
http://www.brenda-enzymes.org/literature.php?e=1.5.1.33&r=484952
9232014- Nice Work
Fifth and Sixth WeekAfter transforming we did expression in 2 L or 4 times the amount we did last round of expression so we would have extra protein or at least a higher concentration. We then harvested the cells through sonication and spindown. Last expression round we were concerned that PTR1 dimerized since the FPLC machine mainly had a 60kDa protein while our protein is 30 kDa. To try and avoid dimerization this round, we had a salt gradient of 4 samples. Tube A had a salt concentration of 500 mM, tube B a salt concentration of 750 mM, tube C a salt concentration of 1000 mM and tube D a salt concentration of 400 mM. The salt should ideally prevent interactions between two protein molecules and prevent dimerization. We then ran and two SDS characterization gels of the highest salt concentration and the lowest salt concentration samples C(900 mM) and D(400mM). Assuming everything is the same, if samples C and D had not dimerized then neither would the other samples with salt concentrations between C and D. After SDS we plan on doing FPLC to further purify our protein and ensure it has not dimerized. We have already started on virtual screening.
As labeled above: 1 kDa is a 1kDa protein ladder.0 Sample 0(cell lysate before induction)1 Sample 1(cell lysate after induction) 2C Sample 2(Soluble fraction)for sample C3D Sample 3(Flow through) for sample D4C Sample 4(Wash) for sample C5C Sample 5(elution 1) for sample C 6C Sample 6(elution2) for sample C3C Sample 3(flow through) for sample C
Fig 4) SDS Page Gel protein characterization for sample C or PTR1 sample with 400 mM NaCl. The banding in elutions 1 and 2 appears to be around 35 kDa which is the right size for PTR1. It does not appear to have dimerized and there appears to be little contaimination. We will run FPLC to further purify. Since neither this sample nor the one in Fig 5 appear to have dimerized it seems likely that sampels A and B will also not have dimerized.
As labeled above: 1 kDa is a 1kDa protein ladder.0 Sample 0(cell lysate before induction)1 Sample 1(cell lysate after induction) 2D Sample 2(Soluble fraction)for sample D3D Sample 3(Flow through) for sample D4D Sample 4(Wash) for sample D5D Sample 5(elution 1) for sample D 6D Sample 6(elution2) for sample D
Fig 3) Kan+ Suc agar plates with BL21 cells transformed with 50 ng of PNIC+PTR1 plasmid.
Fig 2) Kan+ Suc agar plates with BL21 cells transformed with 25 ng of PNIC+PTR1 plasmid.
Fig 1) Kan+ Suc agar plates with BL21 cells transformed with 5 ng of PNIC+PTR1 plasmid.
Due to contaimination in the 4 degree fridge we retransformed BL21 cells with PNIC+PTR1.
Third and Fourth Week of Fall
We grew up the transformed BL21 cells from the second week and performed protein expression. We then used chromatography/binding assay with nickel to further purify the protein and took samples for an SDS page gel.
Fig 1) Nanodrop for elution 1(protein) during purification concentration of 1.4 mg/mL measured at wavelength 280 nM.
Fig 2) SDS-page gel ran for our protein Pteridine reductase from L. Major. We failed to get a sample 0, but the samples are as follow:
Well 3-Cell lysate after induction(sample 1)Well 4-Soluble fraction(sample 2)Well 5-Flow through(sample 3)Well 6-Wash(sample 4)Well 7 -Elution 1(sample 5)Well 8-Elution 2(sample 6)
Well 2:ladderWell 3:Sample 1(cell lysate after induction)Well 4: Sample 2(Soluble fraction)Well 5: Sample 3(Flow through)Well 6: Sample 4(Wash)Well 7:Sample 5(elution 1)Well 8: Sample 6(elution2)The bands were around 30 kDa weight which matched our protein, there didn't appear to be a lot of contaimination but we still ran FPLC just to ensure purity.
Fig 3) FPLC graph results. We took tubes 28-33 and combined them for our sample. It was odd that the size of our sample(30 kDa) didn't match up with the FPLC machine which was around 50~60 kDa.
Fig 4) First attempt at concentrating after FPLC. Nanodroped, total volume of sample was 4mL but too low concentration
Fig 5) Second attempt at concentrating further after Fig 4. Nanodrop concentration, total volume of sample was 1 mL, concentration was too low but could not reasonably concentrate it to a lower volume.
After concentrating the sample further, we stored .5 mL in 20% glycerol and another .5 mL by snap freezing in liquid nitrogen. We will perform an enzyme assay to see which storage works better, hopefully even with low volume and concentration we will be able to get some indication of which works best. We need to begin virtual screening and restart protein expression. The protein has 3 possible intermembrane regions which would explain the low yields we've been getting after FPLC. Next protein expression we are going to use 2 L of LB to grow up the bacteria which will hopefully have a high enough concentration to move forward.
Week 10+11/first and second week of fall
Fig 1) DNA sequence forward for sample B1 using T7 primers
Fig 2) DNA sequence reverse for sample B1 using T7 primers
Fig3) Pairwise alignment of sample B1 and desired DNA sequence(from codon optimized sequence). The N's were the 2% that didn't match, and there were no insertions or deletions. The N's were then confirmed using the chromatograph to be the correct bases. Query= B1 sent in sequence Sbjct=is desired sequence
Fig 4) DNA sequence similarity between B1 and desired DNA sequence.
Fig 5) Nanodrop of one clone sample DNA D1 that had a concentration 56.5 ng/uL(sample was sent to DNA sequencing)
Fig 6) Nanodrop of one clone sample DNA D2 with a concentration of 32.3 ng/uL it was sent of to DNA sequencing
Fig 7) Nanodrop of one clone sample of DNA D3 with concentration of 53.0 ng/uL sent off to DNA sequencing.
Fig 8) Nanodrop of Midipreped B1(pNIC + DNA insert) plasmid and got a concentration of around 58.6 ng/uL.
Fig 10) Transformed BL21 cells with 5 ng clone DNA sample B1 ~1 colony growth
Fig 11) Transformed BL21 cells with 25 ng of clone DNA sample B1 a lot of colony growth.*We nanodroped 7 samples about, 3 samples were high enough in concentration to send to DNA sequencing, but only B1 came back as a positive clone. Need to return to lab to get nanodrop pictures. We midipreped an overnight B1 culture, and used the DNA to transform BL21 cells which we will check on Saturday.Week 9
Fig 2) Plate with two smears, bottom is C and top is D. C had a ratio of 1 accepting vector to 6 PCR product. D had a ratio of 3 accepting vector to 5 PCR product.
Fig 3) Plate with two smears, bottom is E and top is F. E had a ratio of 4 accepting vector to 7 PCR product. F had a ratio of 1 accepting vector to 9 PCR product.
Fig 4) Plate with two smears, bottom is G and top is H. Top had a ratio of 2 accepting vector to 7 PCR product. Bottom had a ratio of 1 accepting vector to 10 PCR product.
Fig 5) Plate with two smears, bottom is I and top is J. A had a ratio of 1 accepting vector to 7 PCR product. B had a ratio of 2 accepting vector to 5 PCR product.
Fig 6) Plate with two smears, bottom is A and top is B. A had a ratio of 1 accepting vector to 7 PCR product. B had a ratio of 2 accepting vector to 5 PCR product.
Fig 7) Master plate C, the numbers and colonies are as follow:
Fig 8) Masterplate D, the numbers and colonies are as follow
*The nanodrop concentrations for the past master plates have been remarkably low as in less than 20 usually around 10. I had about two colonies from master plate B that had concentrations of 23.4 and 24.9 ng/uL which was enough to send to sequencing. Also I realized that I've been eluting with too much Tris buffer. I've been following the sigma aldrich instructions and using 100 uL instead of 50 uL from the modified cloning protocol. That would decrease the concentration of the miniprep. Therefore I want to rerun all of the masterplates I've made thus far. So in the fall I should redo about 30(9[masterplate A not shown]+9[Masterplate B not shown]+9[Master plate C shown above]+3[Masterplate D shown above]). Hopefully one will yield a positive clone.
Week 8
Fig 6) Gel electrophoresis of PCR squared. 1st well is a 1 kb ladder, the rest of the wells are tubes of PCR squared. Wells 2-5 were concentrated to 208.6 ng/uL(shown in fig. 4) and wells 5-7 were concentrated into 103.1ng/uL(shown in fig 5). Worried about contamination getting for using these samples in ligation independent cloning. Since vectors are more likely to accept smaller DNA bands. Using these samples for initial cloning however, and going to make another PCR squared and more plates.
F
Fig 5) nanodrop concentration for PCR cleanup for the target(a different sample than fig 4 as a backup).
Fig 3) 7.21.14 Gel electrophoresis of PNIC digested by BSAI. Two bands show that plasmid was digested properly and can be used as an accepting vector.
Fig 1) 7.20.14 Digested accepting vector for PNIC nanodrop concentration. It is a bit low, but can continue.
Hailey - ok good work. put some dates on these gels (above or below them) Also, don't start the caption with 'this is' - just put 'Agaros Gel of PCR......"Have you started the cloning steps. If not be sure you have enought pNIC-Bsa4. - Dr. B 072114
Fig 1) This is a gel of PCR squared first and last well has the 1 kb ladder. The second well is empty, the rest of the wells have the PCR squared reaction. Wells three and four are not as bright so I did not use them in PCR cleanup. I used wells 5 through 8 in PCR clean-up. The Size is around 900 kb which is the right size for the gene. I need to nanodrop this again next week since I forgot to email myself the graphs but the concentration was around 71 ng/uL for the PCR cleanup.
Fig 2) This a gel of secondary PCR with a temperature gradient. The 1st well has the 1 kb ladder. All of the samples had the same amount of Primary PCR(2 uL), 1 uL of forward primer, 1 uL of reverse primer, 10uL of Reaction buffer, 0.5 uL of Q5 Polymerase(diluted 1:2 from stock) and 5 uL of dNTPs. The protocol differed from the standard Q5 protocol in that the elongation time was extended to 40 seconds. The second well had an anealing temperature of 72 C, the third well 71.6 C, the forth 70.7 C, the fith 67.2C, the sixth 65.6 C, 64.6 C and the seventh 64 C. The brightest band was the fifth well or the well with the annealing temperature of 64.6 C which is the annealing temperature I'll use for PCR squared.
Fig 3) Redid primary PCR for target. The 1st well is the 1 kb ladder, well 2 and 3 are primary PCRs. Since they've formed streaks I used them in Fig 2 for secondary PCR
Also in wet lab we digested PNIC and started growing PNIC bacteria to make an accepting vector. I need to nanodrop it next week in addition to the DNA.
Congrats on your secondary working. Include a virtual gel for your RE Digest so we can compare what it's suppose to be and how it came out. -Grace (7/15/14)= ==
Fig 1) Here is a carry over from last week. I ran this gel on friday for PNIC, the ladder is 100 kb. There are 2 bands in the 2nd and 3rd well
.306 ng of DNA, the third well had about 3.06 ng of DNA and the fourth well about 30.6 ng of DNA.
Fig 2) Here is a picture of a restriction enzyme digest we did of pgbr22. The first well is a 1 kb ladder, 2nd well is uncut plasmid of pgrb22, 3rd well is pgbr22 cut with ECOR1, 4th well is pgbr22 cut with PvuII, and the 5th well is pgbr22 cut with both ECOR1 and Pvu II. The 3rd well has one band, the 4th well 2 bands and the 5th 2 bands bright bands and possibly 3 additional bands which might be contamination. Other than that it matched the virtual gel.
Fig 3) Here is a picture of the successful run of primary PCR. The 1st well is a 1 kb ladder, and wells 4 and 5 are primary PCR attempts. 4 worked, however 5 did not appear to.
Working on secondary PCR hopefully will have full DNA by end of week(goal *cross fingers*)(Ha. Ha. Ha. No.)
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Working on secondary PCR have to remake primary PCR. Remaking oligomix, altered annealing temperature for a gradient between 64-72 C for 8 samples and increased elongation time for 40 seconds based on the Q5 website recommendations for complex sequences.
Week 5Hailey - good protein gel image. Not sure but I think if you run it longer you will get better separation. I can sort of see 2 bands there that correspond to the 2 bands in FPLC. Be sure to indicate what ladder you use in lane 1. For the PCR gels - crop them so that there is not so much black space. You can crop them int he imaging software before ever saving the image. Also use the 'invert' to change all the dark to light. Do another attempt of the pNIC PCR. you should be able to get bands in each lane. Also indicate which ladder you use (1kb or 100 bp). -- Dr. BThis week:virtual lab:
We designed tail primers for our targets and ordered the primers.Wet lab:
ig 1) Ran a protein characterization for Yop H, the gel was weird in that samples ran from one well to another.Well 1-ladder(protein 1kb ladder)Well 2-Cell lystate before induction(sample 0)Well 3-Cell lysate after induction(sample 1)Well 4-Soluble fraction(sample 2)Well 5-Flow through(sample 3)Well 6-Wash(sample 4)Well 7 -Elution 1(sample 5)Well 8-Elution 2(sample 6)
Fig 2) The gel electrophoresis of PCRed pGFP from last week. It's hard to see but the fourth well has one band that is about 400-500 bp based on the ladder. There is only one band so there doesn't appear to be any contamination.
Fig 3) Gel electrophoresis of PCRed PNIC the first well was the ladder the second well had about .306 ng of DNA, the third well had about 3.06 ng of DNA and the fourth well about 30.6 ng of DNA.The ladder was 100 bp.
Fig4) Results from the FPLC, we appear to have two proteins the one to the left is about 40 kD and the one to the right is about 30 kD. The one to the right or the 30 kD protein is probably YopH since we know YopH's size. We are going to concentrate it and freeze it with nitrogen.
Week 4
Hailey, ok good work. For your Midiprepped pGFP - did you run int on an agarose gel to see if it was contaminated at all? For your PCR the two bands look sharp and of the same height (which is good) - be sure to indicate on the caption that it is a PCR gel ( I wasn't sure if your were just running plasmid or it was PCR). Your YopH concentrations for the protein expression look good. We should have enough for FPLC. Screenshots of the notebook - are kind of ok for this, because we really just need the absorbance value and the shape of the curve, but I would recomend against it for any gels because you don't have good resolution to see them well. I am looking forward to seeing your gel aftter SDS-PAGE. Thanks Dr. B
(running pGFP gel this week after I PCR it, ran out of time last week)
Virtual lab:
We analyzed DNA sequences online and designed and plasmid, then we ran virtual digestion on each plasmid.
Wet lab:
Updates for DNA sequencing stuff from last week results and next steps:
Fig1) Icmb resubmitted the pGFP sequence, it is better than the last submission but the blast below doesn't identify it as pGFP.
Fig 2) Blast for pGFP sequence to compare it, but no recognition as pGFP. Going to run a PCR on pGFP and then run it on a gel to make sure there is no contamination.
Fig 3) The chromatography for the pmcherry sequence from last week. The sequence still needs to be rerun.
New wet lab stuff
Fig 4)PCR gel- Pgbr 22 bands. 1st well was 1 kb ladder, 2nd well was tube A( .3ng of DNA, mastermix and primer), 3rd well was tube B(3 ng DNA, primer and mastermix), 4th well was tube C(30 ng DNA, primer and mastermix) and 5th well was control(no DNA, but primers and mastermix). The wells mainly differed in amount of DNA while all the wells had the same amount of mastermix and the same amount of primers(1 ul of forward and 1 ul of reverse primers). Wells 2,3 and 4 should've had one band each. Wells 3 and 4 were successful but Well 2 didn't show any DNA. The control was not supposed to have any bands so it was successful.
This week we purified our YopH protein, here are the results from the nanodrop for elution 1 and elution 2. We made a protein gel and are going to get the results for our protein characterization next week hopefully.
Fig 5) YopH elution 1 nanodrop concentration was 3.7 mg/ml
Fig 6) YopH elution 1 trial 2 nanodrop concentration was 3.68 mg/ml (average for elution 1 was 3.69 mg/ml)
Fig 7) YopH elution 2 trial 1 nanodrop concentration was 0. 82 mg/ml
Fig 8)
YopH elution 2 trial 2 nanodrop concentration was 0.84 mg/ml (average was 0.83mg/ml for elution 2)
Week 3
Hailey - good work. DNA conc should be 106 ng/ul (nanograms per microliter) units.The pGFP sequence looks pretty bad. We need to look at the electropherogram and determine if we think there is contamination. A Nanodrop read won't give you that information for this type of contaminatinon.
Also - run this sample of pGFP on a gel (about 300 ng of it) to verify if there are contaminating bands. (can run it on another gel that you are already doing if you want)
Include your protein expression (mention it and then enter the cell pellet weight as the 'result' so far) - Dr. B
We made LB with kanomyocin to grow bacteria(which we spent a lot of time on this week)
We grew a plasmid this week in lab using DH4alpha bacteria. We purified pGFP and had an averaged nanodrop concentration of 106 ug/uL. Here are the nanodrop results for the DNA.
We also sent the DNA off for verification to ensure we amplified the correct DNA.
Fig 2) There are a lot of N's, which means unknown nucleotides. ICMB is also going to rerun this sequence.
Also for week 1 and 2 the DNA sequencing had a lot of N's throughout the sequence so I resent it in for sequencing. The plasmid I had was pmcherry and I used the M14 reverse primer. Andee has the forward primer on her page.
The next thing we did this week was protein expression. We first grew BL21 bacteria in LB until it reached a specific concentration and then we introduced IPTG so that the bacteria would produce protein. Our protein was YopH. The next day we further purifed the protein and sonicated it. Then we will hopefully do protein expression next week to verify and post the results.
Also we did PCR(polymerase chain reaction) on pGBR22 and will probably run it next week on agarose gel.
Week 1 and 2
This week we did some more virtual screening of proteins that we can target, DNA sequencing, made LB agar and broth plates and transformed bacteria.
I sequenced the DNA primer pmcherry and sent it off.Here is the FASTA for nucleotide sequence for pm cherry using a reverse primer.
I tried to use heat shock to transform DH5alpha cells with plasmid PNIC Bsa4 with a resistance to Kan. The plates are below, I got an invisible bacteria lawn(not really).
Fig 1) Plate with Dh5alpha cells, plasmid Bsa4 with resistance to Kan(kanamycin) grown on an LB and Kan media. There was no growth and the concentration of plasmid is unknown due to a labeling error. The possible concentrations are 1 ng, 5ng and 25 ng of DNA. Grown for 24 hrs in 37 C incubator.
Fig 3) Plate with Dh5alpha cells, plasmid Bsa4 with resistance to Kan(kanamycin) grown on an LB and Kan media. There was no growth and the concentration of plasmid is unknown due to a labeling error. The possible concentrations are 1 ng, 5ng and 25 ng of DNA. Grown for 24 hrs in 37 C incubator.